This proposal focuses on the fundamental question of how cranial neural crest cells migrate to form facial structures during embryonic development. In particular, we will elucidate the role that TACC3 (transforming acidic coiled-coil containing protein 3) plays during craniofacial development. The TACC3 gene maps to a region of the genome that is deleted (or sometimes duplicated) in Wolf-Hirschhorn Syndrome, a complex disorder with a distinctive craniofacial phenotype. While other genes have been implicated in this disorder, it has recently become apparent that these genes are not acting alone, and others must contribute to the syndrome phenotypes. TACC3 has previously been overlooked, likely due to a lack of mechanistic insight into its possible contribution. However, our recent studies of TACC3 have uncovered novel cell biological functions that may directly contribute to the Wolf-Hirschhorn Syndrome phenotype. Specifically, our work suggests that TACC3 is an important microtubule regulator that has a direct effect on cell motility, including migration of cranial neual crest cells. The central hypothesis that we will test in this proposal is that TACC3 dysfunction contributes to Wolf-Hirschhorn syndrome by playing a critical role in cranial neural crest cell migration. We will this with the following aims: Aim 1: Quantify the craniofacial defects following TACC3 manipulation in Xenopus Our preliminary data suggest that craniofacial defects are present when TACC3 levels are manipulated in Xenopus embryos, a useful model for dissecting details of cell biological mechanisms during development. We will test the hypothesis that TACC3 regulates specific craniofacial characteristics that are abnormal in Wolf-Hirschhorn syndrome, by using TACC3 genetic manipulation strategies and cell transplantation techniques, along with morphometric quantification of craniofacial features and analysis of in vivo cranial neural crest cell migration patterns. embryos. Aim 2: Define the effects of TACC3 manipulation on cranial neural crest cell motility. We find that TACC3 manipulation results in cranial neural crest cell motility defects. We will test the hypothesis that TACC3 regulates specific parameters of cranial neural crest cell motility, by measuring multiple parameters of cell motility and focal adhesion turnover after TACC3 manipulation, in cultured cranial neural crest cells of Xenopus and neural crest cells derived from human ES cells. Aim 3: Determine the structural domains required for TACC3 localization in cranial neural crest cells. Our recent work demonstrates that TACC3 localizes to two distinct sub-cellular domains in neural crest cells - microtubule plus- ends and focal adhesions. We will test the hypothesis that different structural domains mediate TACC3 accumulation at microtubule plus-ends versus focal adhesions. This will be accomplished by mapping the domains required for localization using a series of TACC3 deletion constructs. The experiments in this proposal represent an essential first step in establishing whether TACC3 may be a critical player in Wolf- Hirschhorn syndrome specifically and in cranial neural crest cell migration more generally.